Fig. 4: The synergistic effect of echinacoside and tobramycin against P. aeruginosa aggregates in human cells and murine lungs.

a Viability of PA14-infected 3-D A549 cells (as measured with an LDH assay) exposed to different concentrations (25, 50, and 100 μM) of echinacoside, 2 μg/mL tobramycin (2Tob) or the echinacoside/tobramycin combination treatments normalized to untreated/uninfected groups. Red bars highlighted the successful combination treatment that potentiated the efficacy of tobramycin and elevated cell viability. (6 independent experiments with four technical replicates; error bars indicate standard deviation). *p < 0.05; **p < 0.01 (one-way ANOVA, with post-hoc Dunnett’s tests for multiple comparisons between 2 groups). b Representative transmission light micrographs (upper panel) of 3-D A549 cells attached to/detached from collagen-treated carrier beads and fluorescent micrographs (lower panel) of GFP-tagged PA14 attached to A549 cells. Some typical PA14 aggregates attached to A549 cells with strong fluorescent signals are highlighted with white arrows. Scale bar = 400 μm. c The bacterial load in each uninfected lung with empty agar beads, PA14-infected lung, or PA14-infected lung after exposure to 7.2 µg/mouse tobramycin (Tob), 5.2 µg/mouse echinacoside, or the echinacoside/tobramycin combination treatments. The red bar highlighted the successful combination treatment that potentiated the efficacy of tobramycin. Each group contained 5 mice, and 3 technical replicates were performed for CFU counting. Error bars indicate standard deviation, ***p < 0.001 (one-way ANOVA, with post-hoc Dunnett’s tests for multiple comparisons between 2 groups).